A pilot study evaluating non-contact low-frequency ultrasound and underlying molecular mechanism on diabetic foot ulcers

Abstract

Non-contact low-frequency ultrasound (NCLF-US) devices have been increasingly used for the treatment of chronic non-healing wounds. The appropriate dose for NCLF-US is still in debate. The aims of this pilot study were to evaluate the relationship between dose and duration of treatment for subjects with non-healing diabetic foot ulcers (DFUs) and to explore the correlation between wound healing and change of cytokine/proteinase/growth factor profile. This was a prospective randomised clinical study designed to evaluate subjects with non-healing DFUs for 5 weeks receiving standard of care and/or NCLF-US treatment. Subjects were randomly assigned to one of the three groups: application of NCLF-US thrice per week (Group 1), NCLF-US once per week (Group 2) and the control (Group 3) that received no NCLF-US. All subjects received standard wound care plus offloading for a total of 4 weeks. Percent area reduction (PAR) of each wound compared with baseline was evaluated weekly. Profiles of cytokines/proteinase/growth factors in wound fluid and biopsied tissue were quantified to explore the correlation between wound healing and cytokines/growth factor expression. Twelve DFU patients, 2 (16·7%) type 1 and 10 (83·3%) type 2 diabetics, with an average age of 58 ± 10 years and a total of 12 foot ulcers were enrolled. Average ulcer duration was 36·44 ± 24·78 weeks and the average ABI was 0·91 ± 0·06. Group 1 showed significant wound area reduction at weeks 3, 4 and 5 compared with baseline, with the greatest PAR, 86% (P < 0·05); Groups 2 and 3 showed 25% PAR and 39% PAR, respectively, but there were no statistically significant differences between Groups 2 and 3 over time. Biochemical and histological analyses indicated a trend towards reduction of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α and GM-CSF), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and macrophages in response to NCLF-US consistent with wound reduction, when compared with control group subjects. This proof-of-concept pilot study demonstrates that NCLF-US is effective in treating neuropathic diabetic foot ulcers through, at least in part, inhibiting pro-inflammatory cytokines in chronic wound and improving tissue regeneration. Therapeutic application of NFLU, thrice (3) per week, renders the best wound area reduction.

Keywords: Chronic wound; Diabetic foot ulcers; Inflammatory cytokines; Matrix metalloproteinase; Non-contact low frequency ultrasound; Vascular endothelial growth factor.

Figure 1

Change of wound size with the non‐contact low‐frequency ultrasound (NCLF‐US) therapy. Twelve eligible subjects with chronic diabetic foot ulcers were randomised into one of the three groups. NCLF‐US therapy (with Celleration MIST Therapy System 5.0^®) was applied for the treatment of chronic wounds. Wound sizes were calculated by length × width and expressed as percent change from baseline wound size at week 1.

Figure 2

Change of pro‐inflammatory cytokines in wound fluid in response to non‐contact low‐frequency ultrasound (NCLF‐US) therapy. Wound fluids were collected during the study visits. Various molecular markers, including IL‐6, IL‐8, IL‐1b, TNF‐α and GM‐CSF (A–E) were quantified using Luminex 100 multiplex assays. Actual reading values were log‐transformed prior to analysis due to skewed distribution. Results were also expressed as percent change compared with baseline levels of these markers to avoid the discrepancy among baseline values.

Figure 3

Change of matrix metalloproteinases (MMP)‐9 in wound fluid in response to non‐contact low‐frequency ultrasound (NCLF‐US) therapy. Wound fluids were collected during the study visits. MMPs and TIMPs were quantified using Luminex 100 multiplex assays. Actual reading values were log‐transformed prior to analysis due to skewed distribution. Results were also expressed as percent change compared with baseline (week1) levels of these molecular markers (A). Correlation analysis on MMP and wound size was also performed (B).

Figure 4

Change of vascular endothelial growth factor (VEGF) in wound fluid in response to non‐contact low‐frequency ultrasound (NCLF‐US) therapy. Wound fluids were collected during the study visits. VEGF were quantified using Luminex 100 multiplex assays. Actual VEGF reading values were log‐transformed prior to analysis because of skewed distribution. Results were also expressed as percent change compared with baseline (week1) levels of these molecular markers (A). Correlation analysis on VEGF and MMP was also performed (B).

Figure 5

Macrophage infiltration during wound healing in diabetic foot ulcers. Ulcer tissues were obtained during each study visit. Macrophage numbers were quantified as determinants of inflammatory infiltrate (A). CD68 was used as the specific marker of macrophage numbers and fluorescent dye (FITC)‐labelled CD68‐positive cells were counted using a fluorescence microscope (B). Arrows indicate areas of intense CD68+ cell infiltration. Clusters or individual cells are shown with *.